HIV microbicides are designed to be applied topically before sexual intercourse to inactivate the virus and prevent infection. Some of the most promising microbicide candidates have been proteins, but their clinical development and evaluation has been hampered by the lack of available material and/or the prospect of having to manufacture vast quantities of recombinant protein very cheaply. Plant biotechnology offers some potential solutions. Whilst the production of microbicides at agricultural scale is a long term aim, it is likely that the first generation products will emerge from plants grown in containment, under conditions more recognizable as conventional medicine production systems. It has long been established that recombinant proteins can be expressed in all tissues of the plant, including roots. Indeed, some recombinant proteins produced by transgenic plants are actively secreted from the root system in a process known as rhizosecretion. This gives rise to the possibility that transgenic plants could be grown in greenhouses under hydroponic conditions, using a defined culture medium. Moreover, the microbicide product could be harvested from hydroponic culture medium, rather than plant tissue, which would greatly simplify purification, and allow harvest over the lifetime of the plant. Hydroponic cultivation of plants is already a well established technique in the horticultural industry and is also currently used for the production of natural medicinal compounds. The objective of this proposal is to establish a contained hydroponic tobacco plant culture approach for production of two microbicide protein candidates, cyanovirin-N and MAb 4E10, and to develop optimization strategies for growth and production that will deliver previously unavailable protein microbicides at a level to allow clinical evaluation. We will establish production at small commercial scale. In the first (R21) phase of the proposal, we intend to demonstrate feasibility of the approach and have established production driven milestones for entry into the second (R33) phase, in which we will develop manufacturing and purification according to Good Practice regulatory requirements ultimately to deliver protein microbicides for clinical trials. Cyanovirin-N and MAb 4E10 are two of the most promising protein microbicide candidates currently available. However, the clinical development of both has been held back by production difficulties, and their efficacy and safety profiles are still to be determined. This project is aimed at developing a production platform for CV-N, MAb 4E10 and ultimately other recombinant protein microbicides, that will advance these products to human clinical trials.